7-Substituted camptothecin and camptothecin analogs and methods for producing the same

ABSTRACT

Methods of forming camptothecin compounds which are effective anti-tumor compounds are disclosed. These compounds inhibit the enzyme topoisomerase I and may alkylate DNA of the associated topoisomerase I-DNA cleavable complex.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to methods of preparing 7-substitutedcamptothecin compounds and 7-substituted camptothecin analogs.Camptothecin (CPT) and CPT analogs have been reported to inhibit theenzyme topoisomerase I and have in vitro and in vivo anticanceractivity. It has been shown that a large number of substituents can beplaced at C7 of CPT without loss of activity (Redinbo et al., Science279, 1504-1513, 1998).

2. Background of the Invention

Camptothecin (CPT) is a naturally occurring cytotoxic alkaloid which isknown to inhibit the enzyme topoisomerase I and is a potent anti-tumoragent. Camptothecin compounds have the general ring structure shownbelow.

Camptothecin was isolated from the wood and bark of Camptothecaacuminata by Wall et al. (Wall et al., 1966, J. Am. Chem. Soc.,88:3888).

Major synthetic efforts have been directed to derivatizing the B-ring atC7 to improve cytotoxic and in vivo activity.

The cytotoxic activity of camptothecin compounds is believed to arisefrom the ability of these compounds to inhibit both DNA and RNAsynthesis and to cause reversible fragmentation of DNA in mammaliancells. Topoisomerase I relaxes both positively and negativelysupercoiled DNA and has been implicated in various DNA transactions suchas replication, transcription and recombination. The enzyme mechanism isbelieved to involve a transient breakage of one of the two DNA strandsand the formation of a reversible covalent topoisomerase I enzyme-DNAcomplex. Camptothecin interferes with the DNA breakage-reunion reactionby reversibly trapping the enzyme-DNA intermediate termed the “cleavablecomplex.” The cleavable complex assay is a standard test for determiningthe potential and in vivo cytotoxic activity of camptothecin compounds.The high levels of topoisomerase I in several types of human cancer andthe low levels in correspondingly normal tissue provide the basis fortumor treatment with biologically active camptothecin analogs.

U.S. Pat. No. 4,894,456 describes methods of synthesizing camptothecincompounds which act as inhibitors of topoisomerase I and are effectivein the treatment of leukemia (L-1210). U.S. Pat. No. 5,225,404 disclosesmethods of treating colon tumors with camptothecin compounds.

Numerous camptothecin compounds and their use as inhibitors oftopoisomerase I are reported by U.S. Pat. No. 5,053,512; U.S. Pat. No.4,981,968; U.S. Pat. No. 5,049,668; U.S. Pat. No. 5,106,742; U.S. Pat.No. 5,180,722; U.S. Pat. No. 5,244,903; U.S. Pat. No. 5,227,380; U.S.Pat. No. 5,122,606; U.S. Pat. No. 5,122,526; and U.S. Pat. No.5,340,817.

U.S. Pat. No. 4,943,579 discloses the esterification of the hydroxylgroup at the 20-position of camptothecin to form several prodrugs. Thispatent further discloses that the prodrugs are water soluble and areconverted into the parent camptothecin compounds by hydrolysis.

Wall et al. U.S. Pat. Nos. 5,646,159 and 5,916,892 disclose C₂₀ aminoacid esters of CPT compounds.

Wall et al. U.S. Pat. No. 5,932,588 disclose CPT compounds bearing a C7methylene leaving groups at C₇ such as —CH₂L where L is Cl, Br, I,—OSO₂CH₃, —OSO₂C₆H₄—CH₃, etc.

Brangi et al., Cancer Research, 59, 5938-5946 Dec. 1, 1999, report aninvestigation of Camptothecin resistance in cancer cells and report thecompound difluoro-10, 11-methylenedioxy-20(S)-camptothecin and severalC7-substituted compounds.

A need continues to exist, however, for a method of preparing7-substituted camptothecin compounds. Refs: Du et al., Biorg. and Med.Chem. 10, 103-110 (2002); Dallavalle et al., J. Med. Chem. 44, 3264-3274(2001).

The procedure of Sawada et al., Chem. Pharm. Bull. 39, 2574-2580 (1991)for preparing 7-alkyl compounds gives adequate yields for C₁₋₃ alkylcompounds; however, the yields for C₄, C₅, C₆-alkyl rapidly become poor.We have discovered a novel way of preparing a large variety of alkyl andaryl C₇-substituted compounds in excellent yields by the reaction of anorthoaminobenzonitrile or appropriately substitutedorthoaminobenzonitrile with a large variety of organometallic reagentwhich will be described in detail in this application.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a methodof preparing 7-substituted camptothecin compounds in excellent yield andwhich cannot be prepared by Sawada et al. procedure (Sawada et al.,Chem. Pharm. Bull. 39, 2574-2580 (1991)) which will be widely applicableto a large number of 7-substituents.

Another object of the present invention is to provide 7-substitutedcamptothecin compounds which cannot be made by the Sawada et al.procedure which include a variety of 7-substituents like sec-butyl,tert-butyl, cyclopentyl, p-fluorophenyl, p-tolyl,p-trifluoromethylphenyl, etc.

Another object of this invention is to prepare lipophilic camptothecincompounds with various substituents at the 7 position.

Another object of the present invention is to provide a method oftreating leukemia or solid tumors in a mammal in need thereof byadministration of 7-substituted camptothecin compounds.

Another object of the present invention is to provide a method ofinhibiting the enzyme topoisomerase I and/or alkylating DNA ofassociated DNA-topoisomerase I by contacting a DNA-topoisomerase Icomplex with a 7-substituted camptothecin compound.

These and other objects of the present invention are made possible by asynthetic method for the preparation of 7-substituted camptothecincompounds of formula (I) or (II):

where

X is H; NH₂; F; Cl; Br; alkyl; O—C₁₋₁₆ alkyl; NH—C₁₋₆ alkyl; N(C₁₋₆alkyl)₂; or C₁₋₈ alkyl;

or X is -Z—(CH₂)_(a)—N—(C₁₋₆ alkyl)₂ wherein Z is selected from thegroup consisting of O, NH and S, and a is an integer of 2 or 3;

or X is —CH₂NR R³, where (a) R² and R³ are, independently, hydrogen,C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkyl-C₁₋₆ alkyl, C₂₋₆ alkenyl,hydroxy-C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ COR⁴ where R⁴ is hydrogen, C₁₋₆alkyl, perhalo-C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkyl-C₁₋₆ alkyl,C₂₋₆ alkenyl, hydroxy-C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ alkoxy-C₁₋₆ alkyl,or (b) R² and R³ taken together with the nitrogen atom to which they areattached form a saturated 3-7 membered heterocyclic ring which maycontain a O, S or NRs group, where R⁵ is hydrogen, C₁₋₆ alkyl, alkyl,aryl, aryl substituted with one or more groups selected from the groupconsisting of C₁₋₆ alkyl, amino, C₁₋₆ alkylamino, alkyl, C₁₋₆ alkoxy,C₁₋₆ alkoxy-C₁₋₆ alkyl and alkyl, C₁₋₆ alkoxy, aryl, and arylsubstituted with one or more C₁₋₆ alkyl, C₁₋₆ alkyl, or C-6 alkoxy-C₁₋₆alkyl groups;

R is C₁₋₃₀ alkyl, substituted C₁₋₃₀ alkyl, C₁₋₃₀ alkenyl, substitutedC₁₋₃₀ alkenyl, C₁₋₃₀ alkynyl, substituted, C₁₋₃₀ alkynyl, C₃₋₃₀cycloalkyl, substituted C₃₋₃₀ cycloalkyl, C₆₋₁₈ aryl, substituted C₆₋₁₈aryl, C₆₋₁₈ aryalkyl, (C₁₋₃₀ alkyl)₃ silyl, (C₁₋₃₀ alkyl)₃ silyl C₁₋₃₀alkyl,

Y is independently H or F, and

n is an integer of 1 or 2,

and salts thereof comprising:

i) reacting ortho amino cyano aromatic compound of formula (III) or (IV)

with an organometallic reagent R-M and

ii) condensing a resulting product with a 20(S)tricyclic ketone offormula (VII)

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless indicated to the contrary, the term “alkyl” as used herein meansa straight-chain or branched chain alkyl group with 1-30, preferably1-18 carbon atoms, more preferably 1-8 carbon atoms, including methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl,n-pentyl, isopentyl, n-hexyl, n-heptyl, n-octyl, 2-ethylhexyl, n-nonyl,n-decyl, undecyl, dodecyl, myristyl, heptadecyl and octadecyl groups.Unless otherwise indicated, the term “alkyl” also includes C₃₋₃₀cycloalkyl groups such as cyclopropyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl groups.

“Substituted” means substituted with one or more heteroatom(s) and/orhalogens and/or alkyl groups of 1 to 4 carbon atoms and/or alkenyland/or alkynyl groups of 2 to 4 carbon atoms and/or cycloalkyl groups of3 to 7 carbon atoms and/or aryl groups of 6 to 12 carbon atoms and/orheteroaryl groups, and in which the alkyl, alkenyl, alkynyl, cycloalkyl,aryl or heteroaryl group may be further substituted with one or moreheteroatoms. Where their valency permits, heteroatoms may be substitutedeither within the carbon chain or by attachment to it by single ordouble bonds. For example, , —CH₂—CH₂—O—CH₃, CH₃—CH₂—CH₂O—,—CH₂—CH₂—C(═O)—NH₂, CH₃—CH₂—C(O)—NH— and CF₃—CC— all fall within thisdefinition.

Unless indicated to the contrary, the term “aryl” as used herein means acarbocyclic aromatic ring having 6-18 carbon atoms, preferably 6-10carbon atoms in the aromatic ring structure. The aromatic rings may besubstituted by one or more alkyl group, preferably alkyl groups having1-10 carbon atoms. A particularly preferred aryl group is phenyl.

Unless indicated to the contrary, the term “aralkyl” as used hereinmeans a straight-chain or branched chain alkyl group as defined abovefor the term “alkyl” bonded to an aryl group as defined above for theterm “aryl”. Preferred aralkyl groups are benzyl, phenethyl, etc.

The present method may be practiced by condensation of an ortho aminocyano phenyl compound of formula III or IV

where Y is independently H or F and n is an integer of 1 or 2;

X is H, NH₂, F, Cl, Br, O—C₁₋₆ alkyl, S—C₁₋₆ alkyl, NH—C₁₋₆ alkyl,N(C₁₋₆ alkyl)₂, or C₁₋₈ alkyl,

or X is -Z—(CH₂)_(a)—N—(C₁₋₆ alkyl)₂ wherein Z is selected from thegroup consisting of O, NH and S, and a is an integer of 2 or 3,

or X is —CH₂NR²R³, where (a) R² and R³ are, independently, hydrogen,C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkyl-C₁₋₆ alkyl, C₂₋₆ alkenyl,C₁₋₆ alkoxy-C₁₋₆ COR⁴ where R⁴is hydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl,C₃₋₇ cycloalkyl-C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆ alkoxy, C₁₋₆ alkoxy-C₁₋₆alkyl, or (b) R² and R³ taken together with the nitrogen atom to whichthey are attached form a saturated 3-7 membered heterocyclic ring whichmay contain a O, S or NR⁵ group, where R⁵ is hydrogen, C₁₋₆ alkyl, aryl,aryl substituted with one or more groups selected from the groupconsisting of C₁₋₆ alkyl, amino, C₁₋₆ alkylamino, perhalo-C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ alkoxy-C₁₋₆ alkyl and is hydrogen, C₁₋₆ alkyl C₁₋₆alkoxy, aryl, and aryl substituted with one or more C₁₋₆ alkyl, or C₁₋₆alkoxy-C₁₋₆ alkyl groups;

with an organometallic compound R-M.

Compounds of formula I and II may be prepared by conventional methodsknown to those of ordinary skill in the art, without undueexperimentation. For example, compounds of formula (I) and (II) may beprepared by oxidation of a monoprotected diamine, followed by removal ofthe amine protecting group.

Non limiting examples of suitable organometallic compounds arecyclohexylmagnesium halide, allyl magnesium halide, vinyl magnesiumhalide, ethyl magnesium halide, 4-fluorophenyl magnesium halide,isopropenyl magnesium halide, isopropyl magnesium halide, methylmagnesium halide, ethynyl magnesium halide, cyclopentyl magnesiumhalide, phenyl magnesium halide, benzyl magnesium halide, propylmagnesium halide, 1-propynyl magnesium halide, p-tolyl magnesium halide,o-tolyl magnesium halide, 1-trimethylsilymethyl magnesium halide, hexylmagnesium halide, 2-thiophenyl magnesium halide, 4-dimethylaminophenylmagnesium halide, 4-chloro 1-butenyl 2-magnesium halide,p-methoxylbenzyl magnesium halide, methoxymethyl magnesiumhalide,p-trifluoromethylphenyl magnesium halide, and p-chloro phenylmagnesiumhalide. These organometallic reagents may be prepared by conventionalmethods known to those of ordinary skill in the art without undueexperimentation.

The reaction of the organometallic reagent compound with the compound offormula I or formula II, may be accelerated by the addition of acatalyst. Suitable catalysts include, but are not limited to, CuBr,CuCl, or CuI.

The reaction of the organometallic reagent compound with the compound offormula I or formula II, may be conducted in an organic solvent. Themost common solvents used are ethers, preferably tetrahydrofuran,diethyl ether or the like.

The reaction may be conducted at from 0-70° C., preferably in refluxingtetrahydrofuran.

The product of condensation of organometallic reagent compound with thecompound of formula III or formula IV, is an o-amino ketone of structureV or VI

The product o-amino ketone may be isolated and purified or reacteddirectly with the tricyclic ketone (VII) as described below.Condensation with a tricyclic ketone of formula VII yields the7-substituted camptothecin.

Tricyclic ketone of formula VII may be prepared by conventional methodsknown to those of ordinary skill in the art, such as that described byWall et al. U.S. Pat. No. 5,122,526, the relevant portions of which arehereby incorporated by reference.

The condensation with tricyclic ketone (VII) is typically conductedunder acid catalyzed conditions. The dehydration is preferably performedto help drive the condensation reaction.

Condensation of tricylic ketone (VII) with the aminoketone V or VI maybe in a suitable organic solvent such as toluene, benzene, xylene or thelike.

The Friedlander reaction of the orthoaminoketone and the tricycloketonemay be conducted preferably in refluxing toluene.

Camptothecin compounds have an asymmetric carbon atom at the 20-positionmaking two enantiomeric forms, i.e., the (R) and the (S) configurations,possible. This invention includes both enantiomeric forms and anycombinations or mixtures of these forms. The invention also includesother forms of the camptothecin compounds including solvates, hydrates,polymorphs, salts, etc. Particularly preferred compounds arecamptothecin derivatives having the (S) configuration at the20-position.

Throughout the present application many CPT compounds have been definedwith a substituent X as shown in structure I or II where X is definedand many substituents are shown. With the exception of the simplesubstituents where R is C₁₋₅ alkyl (which can be prepared by the methodof Sawada et al. (Chem. Pharm. Bull. 39, 2574-2580 (1999))), the othercompounds recited herein can not be made by conventional methods, butrequire, to the best of the inventors' knowledge, the use of the methodof the present invention. Using the method of Sawada et al, the yield ofalkyl became progressively lower essentially terminating where R is C₅alkyl. All other R substituents shown herein can be made only by thepresent method involving Grignard reactions, especially analogs like7-t-butyl-10,11-methylenedioxy-CPT,7-trimethylsilymethyl-10,11-methylenedioxy-CPT,7-naphthylmethyl-10,11-methylenedioxy-CPT,7-p-fluorophenyl-10,11-methylenedioxy-CPT,7-p-trifluoromethyl-10,11-methylenedioxy-CPT, and7-p-tolyl-10,11-methylenedioxy-CPT. A number of compounds made by thismethod have shown unusual properties. For example, Table 1 gives themethylene chloride solubility of a number of compounds which are made bythe methods described by this patent. Forexample,7-butyl-10-aminocamptothecin made by this procedure isremarkably lipophilic. 10-Aminocamptothecin has a solubility of _(—)0.2mg/ml. However, 7-n-butyl-10-aminocamptothecin has a solubility of 140mg/mL in methylenechloride, unexpectedly making this compound the mostlipophilic camptothecin known. It has been found by several groups thatlipophilic substituents at the 7 position have excellent activity in theinhibition of topoisomerase I and in in vitro and in vivo cancertherapy. TABLE 1 Sobulity in CH₂Cl₂ Compound Solubility (mg/ml)9-Methyl-CPT 0.1 mg/ml 10-Amino-CPT 0.2 mg/ml 10,11-ED-CPT 0.2 mg/mlCamptothecin 0.6 mg/ml 9-Amino-10,11-MD-CPT <0.02 mg/ml 9-Amino-CPT<0.03 mg/ml 10-OH-CPT <0.04 mg/ml 7-Butyl-10-OH-CPT <0.05 mg/ml10,11-MD-CPT <0.008 mg/ml 7-Butyl-9-methyl-CPT 0.18 mg/ml9-Nitro-10,11-MD-CPT 0.25 mg/ml 7-Benzyl-10,11-CPT 1.8 mg/ml7-Benzyl-10,11-MD-CPT 1.8 mg/ml 7-Butyl-10-methoxy-CPT 2 mg/ml7-p-Fluorophenyl-10,11-MD-CPT 2.55 mg/ml 10-Methoxy-CPT 3 mg/ml7-p-Tolyl-10,11-MD-CPT 4.6 mg/ml 7-p-Chlorophenyl-10,11-MD-CPT 6.3 mg/ml7-Butyl-10,11-ED-CPT 7 mg/ml 7-Butyl-10,11-MD-CPT 8.5 mg/ml 7-Butyl-CPT20 mg/ml 7-(sec)Butyl-CPT 33 mg/ml 7-Butyl-10-Amino-CPT 145 mg/ml

References for the increase in cytotoxic potency: Dallavale et al. NovelCytotoxic 7-Aminomethyl and 7-Aminomethyl Derivatives of Camptothecin,Biorg. & Med. Chem. Lett. 11, 291-294 (2001), Bom et al. Novel A, B,E-Ring Modified Camptothecins Displaying High Lipophilicity and MarkedlyImproved Blood Stability, J. Med. Chem. 42, 30i8-3022 (1999), Bom etal., Novel Silotecan 7-Tertbutyldimethylsilyl-10-hydroxycamptothecinDisplays High Lipophilicity, Improved Human Blood Stability, and PotentAnticancer Activity, J. Med. Chem. 43, 3970-3980 (2000).

We have utilized a standard method for ascertaining the lipophilicity ofa number of camptothecin analogs. This involves the solubility inmethylene chloride. This solvent is an excellent solvent for fat-solublecompounds. As can be noted with only one exception, compounds with the7-butyl substituent are considerably more soluble than those withoutthis constituent. Thus camptothecin is soluble only to the extent of 0.6mg/ml whereas 7-butyl-camptothecin has a solubility of 20 mg/ml.10-Amino-camptothecin is soluble only to the extent of 0.2 mg/ml. Veryunexpectedly, 7-butyl-10-amino-camptothecin is very soluble at 145mg/ml. It is also conceivable that camptothecin analogs with 7-pentyl or7-hexyl substituents or 7-cyclopentyl or 7-cyclohexyl substituents willhave increased solubility.

Another compound with special properties is7-p-fluorophenyl-10,11-methylenedioxycamptothecin with an IC₅₀ of 0.692.In contrast, 10,11-methylenedioxy-CPT has an IC₅₀ of 1.24. The7-p-fluorophenyl-10,11-methylenedioxycamptothecin is the most cytotoxiccompound that has ever been made.

Within the scope of the present invention, the lactone ring of thecamptothecin compounds shown above may be opened by alkali metal oralkaline earth metal bases (MOH) for example, sodium hydroxide orcalcium hydroxide to form alkali metal or alkaline earth metal salts ofthe open ring salt form of the camptothecin compounds, illustrated forexample only for the alkylenedioxy compound.

Open ring compounds generally have better solubility in water. The groupM may also be any pharmaceutically acceptable cation, obtained eitherdirectly by ring opening or by cation exchange of a ring open salt.Suitable groups M include Li⁺, Na⁺, K⁺ and Mg⁺².

The C₂₀ OH CPT compounds of the present invention may be prepared byconventional methods known to those of ordinary skill in the art, suchas that described by Wall et al. U.S. Pat. No. 5,122,526, the relevantportions of which are hereby incorporated by reference.

Esterification with an amino acid at C₂₀ is possible by conventionalmethods known to those of ordinary skill in the art. Suitable estersformed at C₂₀ are those described in U.S. Pat. No. 6,268,375, therelevant portions of which are hereby incorporated by reference.Substitution at C₉ with groups such a nitro and amino is also possiblein a manner analogous to that described in the literature.

The compounds of the invention having the group —CH₂-L at C₉ areprepared from known 20(S)—CPT compounds bearing a halogen, for example,a bromine atom, at the C₉ position. The halogen atom can be readilyconverted into the corresponding cyano analog by reaction with CuCN,followed by hydrolysis to form the corresponding carboxy analog. Thecarboxy analog is reduced to the corresponding hydroxy methyl analogwhich can be reacted with Ph₃P-CCl₄ to provide the correspondingchloromethyl analog. The chloromethyl analog can be readily converted tothe bromomethyl and iodomethyl analogs using LiBr or LiI. The remainingcompounds of the invention are prepared from these compounds by reactionwith the corresponding acid chloride, sulfonyl chloride, etc. Thesereactions are well known to one having ordinary skill in this art.

Compounds in which L is Br or I are readily prepared from the compoundin which L is Cl by simple halide exchange employing LiBr or LiI indimethylformamide (DMF) solution (Larock, R. C., Comprehensive OrganicTransformations, VCH Publishers, Inc., p. 337, N.Y. 1989).

C₂₀ esters may be prepared by esterifying the 20-position hydroxyl groupof a camptothecin compound to form an ester containing a water-solublemoiety. Generally, the camptothecin compound is initially suspended inmethylene chloride or other inert solvent, stirred and cooled. To thecooled mixture is added one equivalent of an acid having the formulaHOOC—CH₂—CH₂—NR⁸R⁹, where R⁸ and R⁹ are independently, hydrogen, C₁₋₈alkyl, C(O)—(CH₂)_(m)—NR¹⁰R¹¹, where m is an integer from 1 to 6, or—C(O)CHR¹²NR¹³R¹⁴, where R¹² is the side chain of one of the naturallyoccurring α-amino acids and R¹⁰, R¹¹, R¹³ and R¹⁴ are each independentlyhydrogen or C₁₋₈ alkyl. Suitable side chains R¹² are the side chains ofthe amino acids glycine, α-alanine, β-alanine, valine, leucine,isoleucine, phenylalanine, tyrosine, tryptophan, leucine, arginine,histidine, aspartate, glutamate, asparagine, glutamine, cysteine andmethionine. A particularly useful ester can be prepared from the peptideβ-alanine-lysine which forms a very water-soluble dihydrochloride Salt.One equivalent of dicyclohexylcarbodiimide (DCC) and a catalytic amountof an amine base, preferably a secondary or tertiary amine, are alsoadded to the mixture, which is then stirred to complete the reaction.Any precipitate which forms is removed by filtration and the product isisolated after removal of the solvent.

The free amine(s) may be converted to an acid addition salt by theaddition of a pharmaceutically acceptable acid. Suitable acids includeboth inorganic and organic acids. Suitable addition salts include, butare not limited to hydrochloride, sulfate, phosphate, diphosphate,hydrobromide, nitrate, acetate, malate, maleate, fumarate, tartrate,succinate, citrate, lactate, methanesulfonate, p-toluenesulfonate,palmoate, salicylate and stearate salts. The salts may be purified bycrystallization from a suitable solvent.

The camptothecin compounds are administered in a dose which is effectiveto inhibit the growth of tumors. As used herein, an effective amount ofthe camptothecin compounds is intended to mean an amount of the compoundthat will inhibit the growth of tumors, that is, reduce the site ofgrowing tumors relative to a control in which the tumor is not treatedwith the camptothecin compound. These effective amounts are generallyfrom about 1-60 mg/kg of body weight per week, preferably about 2-20mg/kg per week.

The compounds of the present invention may be administered as apharmaceutical composition containing the camptothecin compound and apharmaceutically acceptable carrier or diluent. The active materials canalso be mixed with other active materials which do not impair thedesired action and/or supplement the desired action. The activematerials according to the present invention can be administered by anyroute, for example, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid or solid form.

For the purposes of parenteral therapeutic administration, the activeingredient may be incorporated into a solution or suspension. Thesolutions or suspensions may also include the following components: asterile diluent such as water for injection, saline solution, fixedoils, polyethylene glycols, glycerine, propylene glycol or othersynthetic solvents; antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. The parenteral preparationcan be enclosed in ampoules, disposable syringes or multiple dose vialsmade of glass or plastic.

Another mode of administration of the compounds of this invention isoral. Oral compositions will generally include an inert diluent or anedible carrier. For the purpose of oral therapeutic administration, theaforesaid compounds may be incorporated with excipients and used in theform of tablets, gelatine capsules, troches, capsules, elixirs,suspensions, syrups, wafers, chewing gums and the like. Compositions maybe prepared according to any method known to the art for the manufactureof pharmaceutical compositions and such compositions may contain one ormore agents selected from the group consisting of sweetening agents,flavoring agents, coloring agents and preserving agents. Tabletscontaining the active ingredient in admixture with nontoxicpharmaceutically acceptable excipients which are suitable formanufacture of tablets are acceptable. These excipients may be, forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate granulating anddisintegrating agents, such as maize starch, or alginic acid; bindingagents, such as starch, gelatin or acacia; and lubricating agents, suchas magnesium stearate, stearic acid or talc. Tablets may be uncoated ormay be coated by known techniques to delay disintegration and adsorptionin the gastrointestinal tract and thereby provide a sustained actionover a longer period. For example, a time delay material such asglyceryl monostearate or glyceryl distearate alone or with a wax may beemployed. Formulations for oral use may also be presented as hardgelatin capsules wherein the active ingredient is mixed with an inertsolid diluent, for example calcium carbonate, calcium phosphate orkaolin, or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, such as peanut oil, liquid paraffinor olive oil.

The tablets, pills, capsules, troches and the like may contain thefollowing ingredients: a binder such as microcrystalline cellulose, gumtragacanth or gelatin; an excipient such as starch or lactose, adisintegrating agent such as alginic acid, Primogel, corn starch and thelike; a lubricant such as magnesium stearate or Sterotes; a glidant suchas colloidal silicon dioxide; and a sweetening agent such as sucrose orsaccharin or flavoring agent such as peppermint, methyl salicylate, ororange flavoring may be added. When the dosage unit form is a capsule,it may contain, in addition to material of the above type, a liquidcarrier such as a fatty oil. Other dosage unit forms may contain othervarious materials which modify the physical form of the dosage unit, forexample, as coatings. Thus tablets or pills may be coated with sugar,shellac, or other enteric coating agents. A syrup may contain, inaddition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors. Materials used inpreparing these various compositions should be pharmaceutically orveterinarially pure and non-toxic in the amounts used.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylethyl cellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethylene oxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol (e.g.,polyoxyethylene sorbitol mono-oleate), or a condensation product ofethylene oxide with a partial ester derived from fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one ormore flavoring agents and one or more sweetening agents, such assucrose, aspartame, saccharin, or sucralose.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oil suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of anantioxidant such as ascorbic acid.

Dispersible powders and granules of the invention suitable forpreparation of an aqueous suspension by the addition of water may beformulated from the active ingredients in admixture with a dispersing,suspending and/or wetting agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose disclosed above. Additional excipients, for example sweetening,flavoring and coloring agents, may also be present.

The pharmaceutical composition of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, suchas olive oil or arachis oil, a mineral oil, such as liquid paraffin, ora mixture of these. Suitable emulsifyng agents include naturallyoccurring gums, such as gum acacia and gum tragacanth, naturallyoccurring phosphatides, such as soybean lecithin, esters or partialesters derived from fatty acids and hexitol anhydrides, such as sorbitanmono-oleate, and condensation products of these partial esters withethylene oxide, such as polyoxyethylene sorbitan mono-oleate. Theemulsion may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, such asglycerol, sorbitol or sucrose. Such formulations may also contain ademulcent, a preservative, a flavoring or a coloring agent.

The pharmaceutical compositions of the invention may be in the form of asterile injectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a nontoxic parenterally acceptable diluent or solvent,such as a solution of 1,3-butanediol. Among the acceptable vehicles andsolvents that may be employed are water and Ringer's solution, anisotonic sodium chloride. In addition, sterile fixed oils mayconventionally be employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid may likewisebe used in the preparation of injectables. Sterilization may beperformed by conventional methods known to those of ordinary skill inthe art such as by aseptic filtration, irradiation or terminalsterilization (e.g. autoclaving).

Aqueous formulations (i.e., oil-in-water emulsions, syrups, elixirs andinjectable preparations) may be formulated to achieve the pH of optimumstability. The determination of the optimum pH may be performed byconventional methods known to those of ordinary skill in the art.Suitable buffers may also be used to maintain the pH of the formulation.

The compounds of this invention may also be administered in the form ofsuppositories for rectal administration of the drug. These compositionscan be prepared by mixing the drug with a suitable nonirritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperatures and will therefore melt in the rectum to release thedrug. Non-limiting examples of such materials are cocoa butter andpolyethylene glycols.

They may also be administered by intranasal, intraocular, intravaginal,and intrarectal routes including suppositories, insufflation, powdersand aerosol formulations.

The compounds of the present invention may also be administered in theform of liposome or microvesicle preparations. Liposomes aremicrovesicles which encapsulate a liquid within lipid or polymericmembranes. Liposomes and methods of preparing liposomes are known andare described, for example, in U.S. Pat. No. 4,452,747, U.S. Pat. No.4,448,765, U.S. Pat. No. 4,837,028, U.S. Pat. No. 4,721,612, U.S. Pat.No. 4,594,241, U.S. Pat. No. 4,302,459 and U.S. Pat. No. 4,186,183. Thedisclosures of these U.S. patents are incorporated herein by reference.Suitable liposome preparations for use in the present invention are alsodescribed in WO-9318749-A1, J-02056431-A and EP-276783-A.

The camptothecin compounds may be used individually to inhibit thegrowth of tumors. Alternatively, combinations of two or morecamptothecin compounds may be used or combinations of one or morecamptothecin compounds with one or more known anti-tumor compounds. Whena camptothecin compound is combined with a conventional anti-tumorcompound, the camptothecin compound will generally be present in anamount ranging from about 1-99 wt. %, preferably, 5-95 wt. % of thecombined amount of camptothecin and conventional anti-tumor compound.The pharmaceutical compositions noted above may contain thesecombinations of compounds together with an acceptable carrier ordiluent.

The ester compounds of the invention may be administered to treatleukemia and solid tumors in mammals, including humans. The esters ofthe present invention are prodrugs which are hydrolyzed to camptothecincompounds demonstrating inhibitory activity on topoisomerase I. Thecamptothecin compounds formed by hydrolysis of the esters of theinvention are also effective in treating leukemia and solid tumors inmammals. Numerous camptothecin compounds have been shown to be effectiveagainst leukemia using the standard L1210 leukemia assay (Wall et al.(1993), Journal of Medicinal Chemistry, 36:2689-2700). High activity ofcamptothecin and camptothecin analogs has also been shown in the P388leukemia assay (Wall (1983), Medical and Pediatric Oncology,11:480A-489A). The latter reference also provides a correlation betweenanti-leukemia activity as determined by the L1210 and the P388 leukemiaassays with efficacy of camptothecin compounds against solid tumors.Compounds reported as active in the leukemia assays also havedemonstrated activity in a number of solid tumors including a colonxenograft, a lung xenograft, a Walker sarcoma and a breast xenograft(Wall (1983), Table IV, page 484 A). Recent studies have confirmed thecorrelation between topoisomerase I inhibitory activity andanti-leukemia/anti-tumor activity of camptothecin compounds (Giovanellaet al. (1989), Science, 246: 1046-1048). The compounds of the presentinvention are particularly effective in the treatment of colon, lung,breast and ovary solid tumors, brain glioma and leukemia. Thesecompounds may also be used to treat malaria.

Different aminoketones used were made by following the general procedureof reacting the nitrile with an appropriate Grignard reagent andhydrolyzing the product.

EXAMPLE 1 2-Amino-4,5-methylenedioxy-phenylbenzylmethanone

To a stirred solution of 1.5 g (10.0 mmol) of2-amino-4,5-methylenedioxy-benzonitrile in THF (40 mL) was added CuBr(50 mg, 0.34 mmol) and benzylmagnesium chloride (40 mL, 1.0 M solutionin Et₂O). The reaction mixture was refluxed for 12 h. After cooling to25° C., H₂O (5 mL) was added followed by 15% H₂SO₄ (15 mL). Afterstirring for 14 h, ether (50 mL) was added. Organic layer was separated.Aqueous layer was extracted with ether (2×50 mL). The combined organiclayer was dried and evaporated. Following chromatography (silica gel,CHCl₃), 1.2 g (52%) of the title compound was obtained. IR (CHCl₃) 1675cm⁻¹ MS m/z 255 (M⁺).

EXAMPLE 2 7-Benzyl-10, 11-MD-20(S)-Camptothecin

A mixture of S-tricyclic ketone (1.0 g, 4.2 mmol),2-amino-4,5-methylenedioxy-phenylbenzylmethanone (1.1 g, 4.3 mmol)acetic acid (1 mL), p-TsOH (50 mg) in toluene (100 mL) was refluxed for15 h. After removing the solvent, the crude product was purified bycolumn chromatography (silica gel, CHCl₃) to yield the product as acream powder (1.33 g, 66%)¹H-NMR (DMSO-d₆) δ 0.89 (t, 3H), 1.91 (m, 2H),4.62 (s, 2H), 5.22 (s, 2H), 5.41 (s, 2H), 6.10 (s, 2H), 6.50 (s, 1H),6.90-7.10 (m, 5H), 7.21 (s, 1H), 8.07 (s, 1H), 8.22 (s, 1H); MS: m/z 483(M+1)⁺.

EXAMPLE 3 7- Trimethylsilylmethyl-10,11 -MD-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use of trimethylsilylmagnesium chloride as the Grignard reagent. ¹H-NMR (DMSO-d₆+CDCl₃) δ0.87 (t, 3H), 1.83 (m, 2H), 2.28 (s, 2H), 5.11 (s, 2H), 5.37 (s, 2H),6.25 (s, 2H), 6.47 (s, 1H), 7.20 (s, 1H), 7.43 (s, 1H), 7.48 (s, 1H). MSm/z 478 (M⁺).

EXAMPLE 4 7-t-Butyl-10, 11-MD-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use of t-butylmagnesiumchloride as the Grignard reagent. ¹H-NMR (DMSO-d₆): δ 0.88 (t, 3H), 1.77(s, 9H), 1.91 (m, 2H), 5.40 (s, 2H), 5.55 (s, 2H), 6.25 (s, 2H), 6.50(s, 1H), 7.22 (s, 1H), 7.44 (s, 1H), 7.56 (s, 1H). MS m/z 448 (M⁺).

EXAMPLE 5 7- Benzyl-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use of benzylmagnesiumchloride and orthoamino benzonitrile. ¹H-NMR (DMSO-d₆) δ 0.89 (t, 3H),1.92 (m, 2H), 4.62 (s, 2H), 5.20 (s, 2H), 5.38 (s, 2H), 6.58 (s, 1H),7.1-7.3 (m, 5H), 7.35 (s, 1H), 7.68 (t, 1H), 7.84 (t, 1H), 8.18 (d, 1H),8.29 (d, 2H). MS m/z 439 (M+1)⁺.

EXAMPLE 6 7-Benzyl-10,11 -DFMD-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use of2-amino-3,4-difluoromethylenedioxybenzonitrile and benzylmagnesiumchloride. ¹H-NMR (DMSO-d₆): δ 0.85 (t, 3H), 1.82 (m, 2H), 4.60 (s, 2H),5.29 (s, 2H), 5.39 (s, 2H), 6.51 (s, 1H), 6.88-7.12 (m, 5H), 8.08 (s,1H), 8.26 (s, 1H). MS m/z 518 (M⁺).

EXAMPLE 7 7 -Benzyl-10-hydroxy-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use of appropriatelyprotected orthoaminobenzonitrile and benzylmagnesium chloride. ¹H-NMR(DMSO-d₆): δ 0.89 (t, 3H), 1.86 (m, 2H), 4.57 (s, 2H), 5.25 (s, 2H),5.41 (s, 2H), 6.5 (s, 1H), 7.05-7.19 (m, 5H), 7.23 (s, 1H), 7.30 (s,1H), 7.36 (d, 1H), 7.92 (d, 1H), 10.29 (s, 1H). MS m/z 454 (M⁺).

EXAMPLE 8 7-p-Fluorophenyl-10,11-MD-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Example 1 and 2 and involving the use of p-fluorophenylmagnesium bromide. ¹H-NMR (DMSO-d₆): δ 0.91 (t, 3H), 1.86 (m, 2H), 5.00(2H), 5.43 (s, 2H), 6.30 (s, 2H), 6.55 (s, 1H), 6.99 (s, 1H), 7.29 (s,1H), 7.52-7.75 (m, 5H. MS: m/z 486 (M⁺).

EXAMPLE 9 7-p-Chlorophenyl-10,11-MD-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use of p-chlorophenylmagnesium bromide. ¹H-NMR (DMSO-d₆): δ 0.93 (t, 3H), 1.89 (m, 2H), 5.08(s, 2H), 5.46 (s, 2H), 6.33 (s, 2H), 6.55 (s, 1H), 7.05 (s, 1H), 7.34(s, 1H), 7.67 (s, 1H), 7.71 (d, 2H), 7.79 (d, 2H). MS: m/z 502 (M⁺).

EXAMPLE 10 7-p-Tolyl-10,11-MD-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use of p-tolyl magnesiumbromide. ¹H-NMR (DMSO-d₆): δ 0.85 (t, 3H), 1.82 (m, 2H), 2.45 (s, 3H),4.94 (s, 2H), 5.39 (s, 2H), 6.24 (s, 2H), 6.49 (s, 1H), 6.97 (s, 1H),7.24 (s, 1H), 7.44 (m, 4H), 7.83 (s, 1H). MS: m/z 482 (M⁺).

EXAMPLE 11 7-Cyclohexyl-10,11-MD-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use of cyclohexylmagnesium bromide. ¹H-NMR (DMSO-d₆+CDCl₃) δ 0.86 (t, 3H), 1.2-1.95 (m,12H), 2.42 (m, 1H), 5.20 (s, 2H), 5.36 (s, 2H), 6.25 (s, 2H), 6.48 (s,1H), 7.15 (s, 1H), 7.42 (s, 1H), 7.66 (s, 1H). MS: nm/z 474 (M⁺).

EXAMPLE 12 7-n-Hexyl-10,11-MD-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use of n-hexyl magnesiumbromide. ¹H-NMR (DMSO-d₆+CDCl₃): δ 0.84-1.89 (m, 15H), 2.63 (m, 2H),5.19 (s, 2H), 5.34 (s, 2H), 6.26 (s, 2H), 6.49 (s, 1H), 7.16 (s, 1H),7.39 (s, 1H), 7.68 (s, 1H). MS: m/z 476 (M⁺).

EXAMPLE 13 7-p-Trifluoromethylphenyl-10,11-MD-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use ofp-trifluoromethylphenyl magnesium bromide. ¹H-NMR (DMSO-d₆+CDCl₃): δ0.89 (t, 3H) 1.87 (m, 2H), 5.08 (s, 2H), 5.41 (s, 2H), 6.29 (s, 2H),6.57 (s, 1H), 7.01 (s, 1H), 7.30 (s, 1H), 7.48-8.06 (m, 5H). MS: m/z 536(M⁺).

EXAMPLE 14 7-n-Butyl-10-Amino-20(S)-Camptothecin

The title compound was prepared following analogous procedures asdescribed in Examples 1 and 2 and involving the use of2,5-diaminobenzonitrile and n-butyl magnesium bromide. ¹H-NMR (DMSO-d₆):δ 0.86 (t, 3H), 0.95 (t, 3H), 1.42-1.86 (m, 6H), 2.97 (m, 2H), 5.19 (s,2H), 5.39 (s, 2H), 5.94 (s, 2H), 6.44 (s, 1H), 7.04 (s, 1H), 7.15 (s,1H), 7.22 (d, 1H), 7.82 (d, 1H). MS: m/z 419 (M⁺).

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

1. A method for the preparation of 7-substituted camptothecin compoundsof formula (I) or (11):

where X is H, NH₂, H, F, Cl, Br, O—C₁₋₆ alkyl, S-C₁₋₆alkyl,NH—C₁₋₆alkyl, N(C₁₋₆alkyl)₂, or C₁₋₈ alkyl, or X is -Z—(CH₂)_(a)—N—(C₁₋₆alkyl)₂ wherein Z is selected from the group consisting of O, NH and S,and a is an integer of 2 or 3, or X is —CH₂NR²R , where (a) R² and R³are, independently, hydrogen, C₁₋₆alkyl, C₃₋₇ cycloalkyl, C₃₋₇cycloalkyl-C₁₋₆alkyl, C₂₋₆ alkenyl, C₁₋₆ alkoxy-C₁₋₆ COR⁴ where R⁴ ishydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkyl-C₁₋₆ alkyl, C₂₋₆alkenyl, C₁₋₆ alkoxy, C₁₋₆ alkoxy-C₁₋₆ alkyl, or (b) R² and R³ takentogether with the nitrogen atom to which they are attached form asaturated 3-7 membered heterocyclic ring which may contain a O, S or NR⁵group, where R⁵ is hydrogen, C₁₋₆ alkyl, alkyl, aryl, aryl substitutedwith one or more groups selected from the group consisting of C₁₋₆alkyl, amino, C₁₋₆ alkylamino, C₁₋₆ alkoxy, C₁₋₆ alkoxy-C₁₋₆ alkyl C₁₋₆alkyl C₁₋₆ alkoxy, aryl, and aryl substituted with one or more C₁₋₆alkyl, or C₁₋₆ alkoxy-C₁₋₆ alkyl groups; R is C₁₋₃₀ alkyl, substitutedC₁₋₃₀ alkyl, C₁₋₃₀ alkenyl, substituted C₁₋₃₀ alkenyl, C₁₋₃₀ alkynyl,substituted, C₁₋₃₀ alkynyl, C₃₋₃₀ cycloalkyl, substituted C₃₋₃₀cycloalkyl, C₆₋₁₈ aryl, substituted C₆₋₁₈ aryl, C₆₋₁₈ aryalkyl, (C₁₋₃₀alkyl)₃ silyl or (C₁₋₃₀ alkyl)₃ silyl C₁₋₃₀ alkyl, Y is independently Hor F, and n is an integer of 1 or 2, and salts thereof comprising: i)reacting an ortho amino cyano aromatic compound of formula (II) or (IV)

with an organometallic reagent R-M and ii) condensing a resultingproduct with a 20(S)tricyclic ketone of formula (VII)


2. The method of claim 1, wherein R-M is selected from the groupconsisting of cyclohexylmagnesium halide, allyl magnesium halide, vinylmagnesium halide, ethyl magnesium halide, 4-fluorophenylmagnesiumhalide, isopropenyl magnesium halide, isopropyl magnesium halide, methylmagnesium halide, ethynyl magnesium halide, cyclopentyl magnesiumhalide, phenyl magnesium halide, benzyl magnesium halide, propylmagnesium halide, 1-propynyl magnesium halide, p-tolyl magnesium halide,o-tolyl magnesium halide, 1-trimethylsilymethyl magnesium halide, hexylmagnesium halide, 2-thiophenyl magnesium halide, 4-dimethylaminophenylmagnesium halide, 4-chloro 1-butenyl 2-magnesium halide,p-methoxylbenzyl magnesium halide, methoxymethyl magnesiumhalide, andp-chloro phenylmagnesium halide, n-butyl magnesium halide, s-butylmagnesium halide, t-butyl magnesium halide andp-trifluoromethylphenylmagnesium halide.
 3. The method of claim 2,wherein said ortho amino cyano aromatic compound is a compound offormula (III), R-M is n-butyl magnesium halide, and R⁷ is n-butyl. 4.The method of claim 2, wherein said ortho amino cyano aromatic compoundis a compound of formula (III), R-M is benzyl magnesium halide, and R⁷is benzyl.
 5. The method of claim 2, wherein said ortho amino cyanoaromatic compound is a compound of formula (III), R-M is p-tolylmagnesium halide, and R⁷ is p-tolyl.
 6. The method of claim 2, whereinsaid ortho amino cyano aromatic compound is a compound of formula (III),R-M is 4-fluorophenyl magnesium halide, and R⁷ is 4-fluorophenyl.
 7. Themethod of claim 2, wherein said ortho amino cyano aromatic compound is acompound of formula (III), R-M is p-chlorophenyl magnesium halide, andR⁷ is p-chlorophenyl.
 8. The method of claim 2, wherein said ortho aminocyano aromatic compound is a compound of formula (III), R-M isp-trifluoromethylphenyl magnesium halide, and R⁷ isp-trifluoromethylphenyl.
 9. The method of claim 2, wherein said orthoamino cyano aromatic compound is a compound of formula (IV), R-M isn-butyl magnesium halide, and R⁷ is n-butyl.
 10. The method of claim 2,wherein said ortho amino cyano aromatic compound is a compound offormula (IV), R-M is s-butyl magnesium halide, and R⁷ is s-butyl. 11.The method of claim 2, wherein said ortho amino cyano aromatic compoundis a compound of formula (IV), R-M is t-butyl magnesium halide, and R⁷is t-butyl.
 12. A 7-substituted camptothecin compound of formula (I) or(II):

wherein X is H, NH₂, H, F, Cl, Br, O—C₁₋₆ alkyl, S—C₁₋₆ alkyl, NH—C₁₋₆alkyl, N(C₁₋₆ alkyl)₂, or C₁₋₈ alkyl, or X is -Z—(CH₂)_(a)—N—(C₁₋₆alkyl)₂ wherein Z is selected from the group consisting of O, NH and S,and a is an integer of 2 or 3, or X is —CH₂NR²R³, where (a) R² and R³are, independently, hydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₃₋₇cycloalkyl-C₁₋₆ alkyl, C₂₋₆ alkenyl, C₁₋₆ alkoxy-C₁₋₆ COR⁴ where R⁴ ishydrogen, C₁₋₆ alkyl, C₃₋₇ cycloalkyl, C₃₋₇ cycloalkyl-C₁₋₆ alkyl, C₂₋₆alkenyl, C₁₋₆ alkoxy, C₁₋₆ alkoxy-C₁₋₆ alkyl, or (b) R² and R³ takentogether with the nitrogen atom to which they are attached form asaturated 3-7 membered heterocyclic ring which may contain a O, S or NR⁵group, where R⁵ is hydrogen, C₁₋₆ alkyl, alkyl, aryl, aryl substitutedwith one or more groups selected from the group consisting of C₁₋₆alkyl, amino, C₁₋₆ alkylamino, C₁₋₆ alkoxy, C₁₋₆ alkoxy-C₁₋₆ alkyl C₁₋₆alkyl C₁₋₆ alkoxy, aryl, and aryl substituted with one or more C₁₋₆alkyl, or C₁₋₆ alkoxy-C₁₋₆ alkyl groups; R is C₁₋₃₀ alkyl, substitutedC₁₋₃₀ alkyl, C₁₋₃₀ alkenyl, substituted C₁₋₃₀ alkenyl, C₁₋₃₀ alkynyl,substituted , C₁₋₃₀ alkynyl, C₃₋₃₀ cycloalkyl, substituted C₃₋₃₀cycloalkyl, C₆₋₁₈ aryl, substituted C₆₋₁₈ aryl, C₆₋₁₈ aryalkyl, (C₁₋₃₀alkyl)₃ silyl or (C₁₋₃₀ alkyl)₃ silyl C₁₋₃₀ alkyl, Y is independently Hor F, and n is an integer of 1 or 2, and salts thereof.
 13. The7-substituted camptothecin compound of claim 12, wherein R is selectedfrom the group consisting of cyclohexyl, allyl, vinyl, 4-fluorophenyl,ethynyl, cyclopentyl, phenyl, benzyl, 1-propynyl, p-tolyl, o-tolyl,1-trimethylsilymethyl, hexyl, 2-thiophenyl, 4-dimethylaminophenyl,2-(4-chloro 1-butenyl), p-methoxylbenzyl, methoxymethyl, p-chlorophenyl, s-butyl, t-butyl, and p-trifluoromethylphenyl.
 14. The7-substituted camptothecin compound of claim 13, wherein R is benzyl.15. The 7-substituted camptothecin compound of claim 13, wherein R isp-tolyl.
 16. The 7-substituted camptothecin compound of claim 13,wherein R is p-fluorophenyl.
 17. The 7-substituted camptothecin compoundof claim 13, wherein R is p-chlorophenyl.
 18. The 7-substitutedcamptothecin compound of claim 13, wherein R is p-trifluoromethylphenyl.19. The 7-substituted camptothecin compound of claim 13, wherein R iss-butyl.
 20. The 7-substituted camptothecin compound of claim 13,wherein R is t-butyl.